The lab began as an independent junior research group on Ion Channel Structure at the Max Planck Institute for Medical Research in Heidelberg, Germany in 1997. In the summer of 2002, the new laboratory moved to the Biochemistry Department of Dartmouth Medical School complete with facilities for protein expression, purification, crystallization and X-ray diffraction. New cryo-electron microscopy equipment is available in the nearby Rippel EM Facility. The lab is affiliated with Dartmouth's programs in Neuroscience, Biophysics, Computational Biology, and Structural Biology and Computational Chemistry. Feel free to stop by and check us out. The former MPI website is here and includes lots of additional information/links/etc.

The goal of our research is to understand the functional characteristics of ion channels and transporters in terms of their molecular structure.  Transmembrane electrochemical gradients underpin a wide variety of essential physiological processes, including photosynthesis and respiration, muscle contraction and nerve signalling.  Highly specialized ion transporters are responsible for establishing and maintaining these gradients, while ion channels are designed to exploit the gradients by selectively and/or temporarily permeabilizing the membrane in response to external stimuli.  Here is an overview of our research projects:

• GLUTAMATE RECEPTORS: The main focus of our research involves the AMPA-receptor subfamily of glutamate receptor ion channels, which are found in the postsynaptic membrane and are responsible for most fast excitatory cell-to-cell communication in the central nervous system. After binding to neurotransmitter released from the presynaptic membrane, they open to conduct cation fluxes that depolarize the membrane and stimulate the receiving cell to fire. The channels then spontaneously close ("desensitize"). The kinetics and magnitude of the current can be fine-tuned to cellular requirements by controlling the nature and identity of the glutamate receptor subunits expressed. We wish to understand this complicated molecular machine at the atomic level. Research projects include:

• Stereochemistry and thermodynamics of ligand binding:

Published crystallographic data from other groups have shown that agonist binding is associated with a Venus-flytrap style cleft closure in the glutamate receptor. How does the interaction and cleft closure proceed? To understand the exact sequence of molecular events, we have combined site-directed mutagenesis with fluorescence spectroscopy to follow the kinetics of agonist association, leading to a model in which rapid docking to one side of the open cleft is followed by cleft closure and trapping of ligand.     

Kinetic Analysis of GluR Ligand Binding Process
Empty ligand-binding domain	DOCKING: Glutamate (green) docks in the cleft, adjacent to  one lobe (blue). This step is fast.	LOCKING: A slower isomerization (cleft closure), locks the agonist in the binding cleft, generating a high-affinity complex

 

To understand the energetics of binding, we have used vibrational spectroscopy and calorimetry to probe individual functional groups' interactions, enabling us to model new interactions within the binding site. This figure represents the electronic bonding configuration of the antagonist DNQX bound to GluR, as identified by vibrational spectroscopy. Band shifts also reveal the strength of protein-ligand interactions, and permit modeling of homologous compounds (e.g. CNQX) based on known structures.

Current focus: How do individual GluR side chains establish the pharmacological specificity of the binding site? Which side chains  are required to communicate conformational changes from the  binding site to the channel gate?

• Conversion of binding energy to mechanical work:

We have expressed and crystallized a glutamate receptor ligand-binding domain that includes peptide linkages right up to the transmembrane domains. The structure reveals additional details about how the force generated by cleft closure is transmitted to the ion pore, opening the channel. In order to expand our understanding of the interdomain and intersubunit conformational interactions within the physiological, oligomeric GluR ion channel, we have developed the expression and purification of the entire extracellular domain of a subunit (80% of the subunit molecular mass). In parallel, we are using electron microscopy to analyze entire receptor complexes.

Current working model of GluR channel activation, showing only two of the four subunits, with the transmembrane domains at the bottom. See Madden, Nat. Rev. Neurosci. 3:91.

Current focus: Crystallization of the GluR extracellular domain and cryo-electron microscopy of intact, solubilized GluR channels, both of which should provide new insight into the oligomeric assembly of the channels, and a structural framework for understanding how conformational changes are communicated between domains and subunits. Comparison of GluR gating to other ligand-gated ion channels (e.g. K⁺-channels)

Electron Microscopic Structure Determination of a Glutamate Receptor
A "tilt-pair" of electron micrographs showing single particles	Random-conical tilt analysis of single- particle views	First 3D reconstruction of a GluR ion channel

• PROTOCADHERINS: A second area of interest concerns the protocadherin family, transmembrane proteins of the cadherin family that are expressed in neurons and are thought to mediate specific transsynaptic contacts required for synapse formation and/or maintenance. The alpha and gamma families have the following genetic structure: polymorphic exons encoding alternative extracellular domains are arranged in a tandem array, and are spliced onto a shared cytoplasmic domain. Our goal is to understand the biochemical basis for signaling by the conserved cytoplasmic domains.

• Research project: Expression and purification of Pcdh cytoplasmic domains. In collaboration with Peter Seeburg's lab (MPI, Heidelberg, Germany), the identification of binding partners that mediate intracellular signals. Expression and purification of these partners for co-crystallization with the Pcdh domains. In addition to providing structural insight into the signaling interactions, this work should enable us to target individual signaling interactions by site-directed mutagenesis and/or molecular mimicry, in order to dissect the physiological role of Pcdh binding.

• CFTR: Finally, we are interested in understanding binding interactions of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel found in the lung and other epithelial tissues. CFTR mutations leading to trafficking and folding defects are the most common source of genetic disease among Caucasians. Our goal is to characterize the interaction of CFTR with binding partners that regulate trafficking and folding processes.

• Research project: Expression and purification of CFTR binding partners identified by our collaborator, Bruce Stanton (Physiology Dept.). Co-crystallization with the CFTR binding epitopes. As for the Pcdh project, we hope to be able to target individual binding interactions to understand and ultimately manipulate CFTR trafficking.

 For more details, see the list of lab publications.



Last update Mar. 8, 2005. This page is maintained by the webmaster